A high affinity HSF-1 binding site in the 5′-untranslated region of the murine tumor necrosis factor-α gene is a transcriptional repressor

Abstract

Tumor necrosis factor-α (TNFα) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40°C) markedly attenuates TNFα expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFα gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5′-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5′-UTR of the TNFα gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFα gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFα promoter (-85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (-1080/+138) TNFα promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions -1080/-845, -533/-196, and -326/-39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal -85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNFα promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5°C. This suggests that HSF-1 might repress TNFα transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.